Project Summary T cell is central to maintaining health. Over-exuberant T cell function leads to debilitating and fatal inflammatory disease. Activated T cells have to grow, exit quiescence, proliferate and differentiate in order to function. Therefore, one of the main goals for T cell research is to reveal the factors critical for the growth, proliferation and differentiation of activated T cells. Genome-wide high-throughput approaches have been quite powerful to reveal genes and pathways critical for T cell function. Most of the T cell high- throughput studies performed in the past used mRNA-based analysis, with the notion that mRNA expression will reflect the protein expression. However, accumulating evidence supports that protein and mRNA expression does not entirely correlate and can be quite different at times. In addition, the post- translational modifications of a protein and the factors a protein associating with can profoundly influence the function of a protein. Thus, to appreciate how the function of activated T cells is controlled, we need to know the protein-networks underlying T cell function in a systemic fashion, the knowledge we grossly lack to date. The recent advance of proteomic technology afforded high-speed, high-throughput, high-sensitivity and high-resolution protein analysis with microgram-scale protein samples. We have established robust experimental system to evaluate functional protein-networks in T cells. This system aided us to successfully identify new factors critical for the function of activated T cells. Encouraged by the results, we set the overarching goal of this study to reveal the protein networks underlying the function of activated T cells. A particular focus of this proposal is to reveal and assess the function of factors whose protein and mRNA are discretely regulated, aiming to identify novel molecular mechanisms that could not be revealed by classic mRNA-based analysis. By combining cutting edge MS/proteomics approach with innovative mouse model and genomic editing/genetic methods, we strive to reach the following research aims: Aim 1: Systemically analyze the protein expression dynamics during T cell growth, quiescence exit, and proliferation. Aim 2: Investigate the differential protein expression and its biological significance for Th cell differentiation. Systemic analysis of the protein expression and interaction in various stages of T cell function will, for the first time, provide an atlas for the protein networks underlying T cell biology. In combination and comparison with mRNA-based genomic analysis, it will establish a molecular map for the normal function of activated T cells. This map will help us to identify new factors and pathways critical for specific T cell functions and to reveal the deregulated factors in T cells in immune diseases including cancer, SCID, inflammation and autoimmunity, aiding the efforts to discover the bio-markers for disease diagnosis and prognosis and to find strategies and drug targets for disease treatment.